Abstract
We have used a fluorescence-activated cytotoxicity protocol, 9-(1'-pyrene)nonanol (P9OH)/UV selection (Morand, O. H., Allen, L.-A. H., Zoeller, R. A., and Raetz, C. R. H. (1990) Biochim. Biophys. Acta 1034, 132-141), to isolate a series of plasmalogen-deficient mutants in a murine, macrophage-like cell line, RAW 264.7. Three of these mutants, RAW.7, RAW.12, and RAW.108, displayed varying degrees of plasmalogen deficiency (48, 17, and 14% of wild-type levels, respectively), and all three mutants were deficient in peroxisomal dihydroxyacetone phosphate (DHAP) acyltransferase activity (5% of wild-type). Unlike previously described Chinese hamster ovary (CHO) cell mutants, the RAW mutants contained intact, functional, peroxisomes and normal levels of alkyl-DHAP synthase activity, a peroxisomal, membrane-bound enzyme. In RAW.7 and RAW.108 cells, the loss of peroxisomal DHAP acyltransferase is the primary lesion. RAW.12 displayed not only a deficiency in the DHAP acyltransferase activity, but also displayed a second lesion in the biosynthetic pathway, a deficiency in delta 1'-desaturase activity (plasmanylethanolamine desaturase, EC 1.14.99.19), the final step in plasmenylethanolamine biosynthesis. The deficiencies expressed in the mutants represent unique lesions in plasmalogen biosynthesis. Since the RAW cell line is a macrophage-like responsive cell line, these mutants can be used to examine the role of plasmalogens in cellular functions such as arachidonic acid metabolism, prostaglandin synthesis, protein secretion, and signal transduction.
Highlights
From the $Department of Biophysics, Boston University School of Medicine, Boston, Massachusetts 02118, the YDepartment of Pediatrics and Human Genetics, Medical College of Virginia, Richmond, Virginia 23298, the11Department of Biochemistry, University of Michigan School of Medicine, Ann Arbor, Michigan 48109, **The Kennedy Institutefor Handicapped Children, Baltimore, Maryland 21205, and the $$Department of Cell Biology and Anatomy, The Mount SinaiSchool of Medicine, New York, New York 10029
Unlike pre- Macrophages show a similar profile, not to as great viously described Chinese hamster ovary (CHO) cell anextent [2].Humanheart muscle is unusual in thatit mutants,the RAW mutantscontainedintact,funccontains very high levels of the plasmalogen form of choline tional, peroxisomes and normal levelsof alkyl-DHAP head group species [1, 2]
We’ve chosen to isolate animal cell lines that are deficient in plasmalogens and determine if any cellular processes are affected
Summary
P9OH-containing medium was removed and replaced with 15 ml of P9OH-free medium. The cells were incubated at 37"C for another5h andthen irradiated for 5 min with long-wavelength ultraviolet light. Samples were developed for 5 cm in chlorolike cell line, RAW264.7 (ATCC TIB71), were obtained from the form:methanol:acetic acid:water (25:15:3:1.5) This separated the American Type Culture Collection. The radioactive products were experiments, selections, and mutageneses, RAW cells were plated out localized by fluorography at -80 "C for 6 days after spraying the TLC in tissue culture dishes (to which theyadhered). 3.0 sample was hydrolyzed by incubation in 0.1 N NaOH in chloro- pCi of [2,3-'H]phytanic acid was added to the medium in 5 p1 of form:methanol (1:4) for 1 h a t 40 "C, completely deacylating phos- ethanol (final phytanate concentratioonf 50 nM) in each culture dish, phatidylethanolamine and removing the acyl group from the 2-posi- and cells were incubated for 24 h a t 37 "C.
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